PROJECT SUMMARY There is a critical need for animal models that more closely recapitulate the post-rehabilitation challenges of patients with substance use disorder (SUD). SUD is a chronic disorder characterized by long- term vulnerability to relapse, even after treatment. Following rehabilitation, patients encounter increased drug availability and social stress, which often trigger relapse and accidental overdose. This Mentored Research Career Development Award (K01) seeks support to investigate the molecular mechanisms underlying social stress-potentiated methamphetamine (meth) relapse in a rodent model that is highly relevant to human SUD. The proposed research will provide the candidate with skills in molecular techniques needed to support long- term research career goals. These techniques include small RNA-sequencing, mass spectrometry and miRNA and proteomics data analysis. The central hypothesis of the proposal is that microRNAs (miRNAs) play a key role in the strong drug craving triggered by social stress and meth availability. Because human studies show that the prefrontal cortex (PFC) inhibits drug craving by decreasing conditioned emotional responding and by enhancing cognitive function needed to cope with stress, this brain region will be investigated. Specifically, this proposal will investigate whether aberrant miRNA and protein changes in the PFC mediate social stress- potentiated relapse. Small RNA-sequencing will be used to identify the candidate miRNAs and their protein targets involved in relapse. Although it is currently unknown how circulating miRNAs reflect complex processes in the brain, the miRNA changes in blood associated with relapse will also be determined. The miRNAs in blood will be correlated to the miRNA levels in the PFC as well as to the magnitude of drug seeking in the rodent relapse model. Proteomics analysis with mass spectrometry will determine the protein changes in the PFC that are associated with social stress-potentiated meth seeking. The functional impact of the miRNA and protein changes observed will be assessed by in vivo manipulations. Specifically, miRNA modulators and small molecule effectors will be injected into the PFC to both block and mimic the potentiation of relapse by social stress. Because miRNAs are stable in human blood and brain, this proposal has the potential to identify biomarkers for relapse that can be translated to human studies and used for assessing efficacy of addiction treatment. Furthermore, the optimization of small molecule effectors utilized in this study could result in therapeutics that restore PFC functioning. Proper PFC functioning would enable successful coping with stress and inhibition of drug craving, thereby preventing relapse to SUD.